Methods for measuring target analytes in biological samples, including bodily fluids (e.g., blood, urine, nasal washes), environmental samples, and bioprocessing samples, often require a combination of biological sample preparation followed by some specific detection assay. Analytes, such as proteins, nucleic acids and cells in biological samples, are typically a dilute component in a complex fluid or solid milieu.
Nucleic acids, such as ribonucleic acid (“RNA”) and deoxyribonucleic acid (“DNA”), are particularly useful target analytes in biological assays. For example, for influenza virus detection, the target analyte may consist of RNA contained within dilute viral particles in a nasal swab. In order to prepare the biological sample, for instance, the specimen must be released from the swab, nasal mucoid matrix must be broken down and viral particles must be opened while target RNA are protected from degrading enzymes. Similar processing steps are required for nucleic acid target analytes in other biological matrices, including bodily fluids or tissues, environmental samples, forensic samples, etc. After performing the appropriate sample preparation, many advanced nucleic acid detection methods require amplification of the target analytes through methods such as polymerase chain reaction (“PCR”), nucleic acid sequence-based amplification (“NASBA”), transcription-mediated amplification (“TMA”), loop-mediated isothermal amplification (“LAMP”) or other enzymatic amplification techniques. All of these methods have varying degrees of sensitivity to contaminants in the target matrix, therefore careful sample preparation is required. Finally, amplification is typically coupled with some type of signal transduction in order to measure the amplified product.
Other useful target analytes include peptides, antigens, antibodies, and other proteins. Often these targets are extremely dilute (e.g., antigen concentrations of picograms per milliliter of blood) in a complex matrix containing debris, a variety of cell types, and a large background of proteins that can be in concentrations many orders of magnitude higher than the target analyte. Common approaches to the detection of peptide or protein targets are variations of the sandwich immunoassay, which uses antibodies with specific affinity to the target analyte to selectively immobilize and detect the target. Signal transduction in immunoassays is often based on antibodies labeled with some signal transduction means, such as enzymes used to drive color changes in the enzyme-linked immunosorbent assay (“ELISA”), fluorescent labels used in the fluorescence immunoassay (“FIA”), chemiluminescence and radioactive labels. Particle agglutination assays and immunochromatographic assays are examples of immunoassays based on particle assembly to yield a visible signal. In histology, fluorescence microscopy and flow cytometry applications, analysis of cell populations also typically requires an immunostaining step, where labeled analyte-specific antibodies are used to colorimetrically or fluorescently label the target analytes. Magnetic particles can also be used as labels for magnetic signal transduction.
Whole cells (e.g., mammalian, plant, or bacteria) and viral particles define another class of target analyte. Again, target analyte cells or particles are frequently found at low concentration in complex sample milieu. For example, clinically relevant bacterial concentrations in blood are 1 to 10 colony forming units per milliliter. Extensive and complex sample preparation and labeling are typically required for detection of cellular targets.
Functionalized particles, including microspheres, beads and nanoparticles, have been used for numerous biological assay applications. Several approaches are highlighted below.
Particle-Based Sample Preparation: Functionalized magnetic particles and beads have been used in the context of biological sample cleanup, concentration and separation. Magnetic particles are commercially available in a range of sizes, carrier matrices (e.g., polymer, silica), designs (e.g., core-shell, embedded iron oxide nanoparticles) and surface chemistries. Magnetic particles enable sample manipulation without expensive or complex equipment requirements. Non-magnetic particles are also used in biological sample preparation. One example is the use of silica particles in the presence of chaotropic buffers to selectively bind nucleic acids.
Particle-Based Detection: Particles can be used to provide detection or signal transduction in biological assays. Exemplary methods include latex agglutination assays, immunochromatographic assays, light scattering assays, and fluorescent particle assays. Agglutination assays are simple, visually-read assays, in which the presence of a target analyte causes agglutination or flocculation of functionalized latex particles. Lateral flow assays and other immunochromatographic methods are also typically visually-read assays, in which particles with specific binding groups (e.g., antibodies) migrate through porous material and, in the presence of the target analyte, accumulate on a line or spot in the porous material where specific binding groups have been immobilized. Numerous particle types (e.g., colored latex, gold, and selenium colloids) are used in immunochromatographic assays. The main disadvantages of the latex agglutination and immunochromatographic approaches are limited sensitivity and limited multiplexing ability. The visual read also renders these techniques qualitative and subjective.
Another particle-based approach to target analyte detection is the use of fluorescently-labeled particles to provide signal transduction in biological assays. Polymer and glass particles containing fluorescent dyes and other luminophores such as lanthanide chelates are commercially available (e.g., Molecular Probes/Invitrogen, Thermo Scientific) and are supplied with surface reactive groups for performing further functionalization. Fluorescent particles have been used in the context of planar waveguide-based detection, and multiple analyte detection methods, based on multiplexed measurement of different fluorescently labeled particles, have been demonstrated (see U.S. patent application Ser. No. 12/617,535, by Moll et al., entitled WAVEGUIDE WITH INTEGRATED LENS and filed 12 Nov. 2009, which is incorporated herein by reference in its entirety). Light scattering particles have also been employed for analyte detection, including light scattering particles bound at planar waveguide surfaces.
Field-Assisted Particulate Assays: Mass transport represents a serious limitation in practical heterogeneous assays performed at solid surfaces. This limitation is particularly important in low volume liquid systems where convective mixing is limited. Suggested methods to overcome mass transport limitations include electrophoretic approaches for concentration and detection of nucleic acids, proteins, and whole cells, and methods that use magnetic particle labels.
Dual-Particle Approaches: Several dual-particle approaches have been described, such as an approach in which latex particle pairs are formed in the presence of a target analyte, enabling proximity-based signal generation via a donor-acceptor oxygen channeling mechanism. Additionally, a system for detection of nucleic acid sequences has been described, which utilizes a magnetic particle with a target-specific oligonucleotide sequence and a dye-encapsulated liposome also with a target-specific oligonucleotide sequence. The particle-liposome combination is used as a sensor for specific RNA targets. In a set of approaches collectively referred to as ‘biobarcode’ assays, a large number of copies of a barcode sequence molecule are generated in the presence of an analyte. Alternatively, self-calibrating assays utilize particle complexes and dual wavelength detection.
A variety of useful particle-based separation and purification methods are available for processing biological samples for subsequent detection assays. The particle-based systems provide a method of signal transduction, and can serve as a detection mode in different biological assay formats. Most of these approaches, however, typically require multiple sample preparation and analyte detection steps with extensive user or machine interventions.